The invention concerns antibodies against the tumor-associated antigen CD33 and their use for immunotargeting of CD33-positive cells. The antibodies according to the invention are suitable for use in the medical field, in pharmacy and in biomedical research.
Cancer (malignant neoplasia) is a class of illnesses in which a group of cells is characterized by uncontrolled cell division, by invasion into and destruction of adjoining tissues and sometimes also by metastasis. These malignant properties of cancer differentiate it from benign tumors which, by their growth, displace surrounding tissue but do not infiltrate it and do not metastasize. Most cancer types form tumors, some do not, such as leukemia that affects the hematopoietic system. Cancer in the context of the invention refers to malignant tumors as well as to hemoblastoses.
In cancer cells, the adjustment of growth, division, and destruction in the united cell structure is not functioning. They are generated when certain genes change, these changes are no longer repaired, leading to non-functionality of certain gene regions, so that the cancer cells stimulate themselves to undergo division and ignore growth-inhibiting signals of the cell environment.
In principle, the immune system attempts to fight the uncontrolled growth of the cells. Problematic in this context is the fact that cancer cells are similar to normal body cells in many respects because they have evolved from them. Therefore, the defense measures of the immune system are usually not sufficient in order to control growth of the tumor.
In case of acute myeloid leukemia (AML), the myelopoiesis is affected, i.e., the part of the hematopoietic system that is responsible for the formation of granulocytes and monocytes. The myeloblasts represent in hematopoiesis an immature precursor of the myeloid white blood cells. In AML, several genetic modifications that concern the genes responsible for cell division result in a single myeloblast for formation of a cell that remains in the immature state and proliferates massively. This proliferation of the immature precursors in the bone marrow and also in the blood characterizes AML.
The modifications may be realized at several positions of the cell cycle so that AML has different phenotypic, genotypic and clinical properties. Modern classification of AML is based on the thesis that the properties and behavior of the tumor cells depend on at which stage of the cell cycle the proliferation was stopped. In many AML patients certain specific cytogenetic distinctive features can be determined which are often of prognostic significance. The genetic modifications code for abnormal fusion proteins, mostly transcription factors, causing the uncontrolled proliferation.
Tumor cells differ from healthy cells by the expression of tumor antigens, i.e., of proteins that are expressed only by the tumor cells. They are generated as a result of the modified genome in the cancer cells, i.e., by modified gene expression. Tumor antigens are located on the external cell membrane of the tumor cells, in the cell plasma, and in the cell nucleus. The tumor antigens as a target structure are the basis for most of the concepts of cancer immunotherapy. Currently, more than 2,000 tumor antigens are known.
Ideal as a target for the cancer therapy are tumor-specific antigens (TSA). i.e., antigens produced only by cancer cells but not by healthy cells. Most tumor antigens are however not tumor-specific but tumor-associated (TAA), i.e., they are also expressed by healthy cells. However, in many tumors the tumor antigens are overexpressed. By mutations in the genome, structural changes in the protein sequence can occur also. Many tumor antigens occur only for certain tumor types and, in this connection, often only in certain situations.
Tumor antigens that are present in case of AML comprise inter alia TAAs that are associated with a series of further tumor illnesses, for example, telomerase reverse transcriptase, Wilm's tumor 1 (WT1)-protein, and survivin. Moreover, there are also TAAs known that are associated with other leukemia illnesses such as CD168 and M phase phosphoprotein. Moreover, there are TAAs found in particular in connection with AML, such as CD33, CD45 and mHAgs (minor histocompatibility antigen).
CD33 is a glycosylated transmembrane protein of 364 amino acids and belongs to the family of sialic acid binding Ig-related lectins (SIGLECs). It is expressed also in early hematopoietic precursor cells, myelomonocytic precursors, and myeloid cells while it is not present on normal pluripotent bone marrow stem cells. Approximately 85-90% of AML patients are positive with regard to CD33. Therefore, CD33 is a particularly attractive target for immunotherapy and is already used as a target in a targeted immunotherapy.
The conventional AML therapy is a chemotherapy for induction of remission; subsequently, this may be followed by several chemotherapies or transplantation of hematopoietic stem cells. In the United States, a monoclonal antibody conjugated with a cytostatic agent, gemtuzumab ozogamicin, was approved in the year 2000 for patients with recurrent AML who are no candidates for the standard chemotherapy.
Antibodies are proteins of the class of globulins which in vertebrates are formed in response to certain substances, i.e., antigens. In combating cancer cells, the body's own polyclonal antibodies only play a very minor role. This results from the fact that most tumor cells do not present sufficiently strongly modified antigens that are recognized as foreign by the body's own immune system so that the body's own antibodies do not bind in a sufficient number to the tumor cells. Monoclonal antibodies are with respect to their structure completely identical and are specific to only one epitope of an antigen.
For cancer therapy, the employed monoclonal antibodies act almost exclusively by the antibody-dependent cell-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC). In this connection, antibodies recruit cytotoxic effector cells, such as NK cells, macrophages, lymphocytes or granulocytes that themselves have no antigen specificity, to the tumor cell by means of their Fc region. The direct damage of the cell by bound monoclonal antibodies, such as by triggering certain intracellular signal cascades that initiate apoptosis by cross-linking of the tumor antigens as a result of antibody binding, is a rare exception.
In order to reinforce the effect of monoclonal antibodies in cancer illnesses, different strategies of drug targeting have been developed in order to utilize the antibodies as carriers for more potent active ingredients, so-called immunoconjugates or antibody derivatives. For this purpose, active ingredients such as radionuclides, toxins (for example, the diphtheria toxin), cytokines or also cytostatic agents were bonded (conjugated) to the corresponding antibodies.
In this connection, it is in principle possible to bind highly potent active ingredients that, in case of free systemic administration would cause intolerable side effects as a result of their high toxicity, to an antibody. The antibody derivatives are comprised of three components: an antibody or antibody fragment, an active ingredient, and a linking member (linker) between antibody and active ingredient.
The first clinically tested antibody directed against CD33 is M195. The humanized variant of this antibody of the isotype IgGI, also known by the name lintuzumab, was tested in clinical studies wherein in phase III no advantage of lintuzumab in combination with chemotherapy was however determined when compared to chemotherapy alone (E. J. Feldman et. al., Phase III Randomized Multicenter Study of a Humanized Anti-CD33 Monoclonal Antibody, Lintuzumab, in Combination With Chemotherapy, Versus Chemotherapy Alone in Patients With Refractory or First-Relapsed Acute Myeloid Leukemia. J. Clin. Oncol. 23 (18) 2005, pp. 4110-4116).
Gemtuzumab ozogamicin is an immunoconjugate of the monoclonal antibody specific to CD33 of the type IgG4 that has been used in the USA for treatment of AML until 2010 but has been taken off the market in the meantime. It is comprised of a humanized monoclonal antibody against CD33 which is conjugated with a cytostatic agent, calicheamicin. The action mechanism is based on the endocytosis of the antibody derivative so that the cytostatic agent coupled thereto develops its toxic action after absorption in the cell. Gemtuzumab ozogamicin was not approved in Europe because the efficacy was not satisfactorily proven in the eyes of the European Medicines Agency EMEA.
The following table shows the amino acid sequences of the CDRs (complementary determining regions, the antigen binding sites of an antibody) of the anti-CD33 antibodies disclosed in the corresponding documents.
SEQSEQSEQDocumentVH CDR1ID NO.VH CDR2ID NO.VH CDR3ID NO.U.S. Pat. No. 7,557,189SYYIH37VIYPGNDDISYNQKFXG38EVRLRYFDV39U.S. Pat. No. 5,730,982DYNMH43YIYPYNGGTGYNQKFKS44GRPAMDY45CN101210048DYNMY49YIDPYKGGTIYNQKFKG50QMITAYYFDY51VL CDR1VL CDR2VL CDR3U.S. Pat. No. 7,557,189KSSQSVFFSSSQKNYLA40WASTRES41HQYLSSRT42U.S. Pat. No. 5,730,982RASESVDNYGISFMN46ASNQGS47QQSKEVPWT48CN101210048KASQDINKYIA52TSTLQP53LQYDNLLT54
A general problem for the therapeutic effectiveness of the monoclonal antibodies is the binding behavior of the antibodies on the cancer cells, i.e., the affinity of the antibodies. Even in cancer cells that present sufficient tumor antigens, the binding rate is often not satisfactorily high. With a molecular mass of approximately 150 kDa, antibodies are moreover generally limited in regard to their tissue migration. In this case, antibody fragments, such as Fab, F(ab)2, or scFv (single chain variable fragments), have significant advantages as a result of their significantly reduced size.
Bispecific antibodies, i.e., antibody derivatives as components of two different monoclonal antibodies, provide new possibilities for therapy concepts in cancer immunotherapy.
Quadroma are bispecific antibodies of the first generation and are comprised each of a heavy chain and a light chain of two different monoclonal antibodies. The two arms of the antibody in this connection are specific to different antigens, respectively. The Fc region is formed commonly of the two heavy chains of the antibodies. By this configuration it is, for example, possible to place the paratope of an antibody specific to the tumor antigen and the paratope of another antibody specific to a lymphocyte antigen onto one arm each of the bispecific antibody. It is thus possible to form a three-cell complex that results from the respective different cells bonded to the paratopes and the effector cell bonded to the Fc region. In this connection, generally an improved activation of the body's own immune cells relative to the tumor cells is achieved.
Bispecific antibodies of the newer generation are comprised of two different scFv fragments. They are connected to each other by a linker. For example, a bispecific antibody can bind with an scFv to tumor cells and with the other scFv to effector cells.
When a paratope is specific to T cells, these cells can be activated also. This is not possible with normal monoclonal antibodies because T cells have no Fc receptor. Bispecific antibodies have moreover a higher cytotoxic potential. They bind also to antigens that are expressed relatively weakly.
Up to now, no bispecific antibodies for clinical use in humans have been approved.
Bispecific antibodies are known in which an scFv binds to the CD3 complex on T cells; these are referred to also as BiTE (bispecific T cell engager) (P. A. Baeuerle et. al., BiTE: Teaching antibodies to engage T cells for cancer therapy. Curr Opin Mol Ther 11, 2009, pp. 22-30).
At the moment, two different BiTE antibodies are in clinical studies. Blinatumomab, an antibody that is specific to CD3 and CD19, is tested in patients in late phases of non-Hodgkin lymphoma and in patients with acute lymphoblast leukemia of the B cell series (B-ALL). MT110 is an antibody that is specific to CD3 and EpCAM (epithelial cell adhesion molecule) and is tested in patients with bronchial carcinoma and patients with gastrointestinal cancer illnesses (R. Bargou en. al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 321, 2008, pp. 974-977; K. Brischwein et. al., MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Mol Immunol 43, 2006, pp. 1129-1143).
Not all monoclonal antibodies are suitable in the form of scFv fragments or for the construction of bispecific constructs. In this connection, in particular the affinity of the antibodies is decisive that is determined by the variable regions. Only antibodies that have an especially high affinity are suitable as scFv fragments because binding to the respective antigen is realized only with one pair of variable regions of the heavy and light chains, in contrast to complete IgG antibodies that have two pairs of variable regions of the heavy and light chains.
For the treatment of AML and other carcinomas, there is the need for a new therapeutic concept. Antibodies that are currently known have an affinity that is too low for tumor-associated antigens such as CD33 in order to be usable therapeutically. Therefore, there is a need for the development of specific antibodies for tumor-associated antigens, for example, CD33, that have a high affinity and are therefore suitable for use in cancer therapy.
Object of the invention is therefore providing new anti-CD33 antibodies in particular with a high affinity to CD33 that enables use of the antibodies as recombinant fragments for immunotargeting.